Low L-selectin as a risk factor for PML

BACKGROUND: The purpose of this research was to validate the low expression of L-selectin (CD62L) in natalizumab (NTZ)-treated patients. CD62L is involved in rolling and transmigration of leukocyte cells. A correlation between CD62LCD4(+) T cells low expression and progressive multifocal leukoencephalopathy (PML) development has been suggested in multiple sclerosis (MS) patients treated with NTZ. METHODS: We performed a flow cytometric analysis on peripheral blood mononuclear cells (PBMC); we collected from 23 healthy donors and 225 MS patients: untreated (n = 19) or treated with NTZ (n = 113), interferon-beta (n = 26), glatiramer acetate (n = 26), fingolimod (n = 23) and rituximab (n = 18). We have also analysed two PML/IRIS (immune reconstitution inflammatory syndrome) patients and four longitudinal samples of a NTZ-treated patients before and during the development of a clinical asymptomatic magnetic resonance imaging (MRI) lesion confirmed as PML by cerebrospinal fluid (CSF) examination. Thirty-five NTZ-treated patients were studied longitudinally with three samples taken 4 months apart. RESULTS: The NTZ-treated patients showed a lower percentage of CD62L (33.68 %, n = 113) than first-line treated patients (44.24 %, n = 52, p = 0.0004). NTZ effect was already clear during the first year of treatment (34.68 %; p = 0.0184); it persisted in the following years and disappeared after drug withdrawal (44.08 %). Three percent of longitudinally analysed patients showed a percentage of CD62LCD4(+) T cells under a hypothetical threshold and one patient with asymptomatic PML belongs to a group which expressed low percentage of CD62LCD4(+) T cells. CONCLUSIONS: Our research confirms that NTZ has a specific effect on CD62LCD4(+) T cells consisting in decreasing of the number of positive cells. The low level of CD62L found in a clinically asymptomatic PML patient strengthens its potential usefulness as a biomarker of high PML risk in NTZ-treated patients. A larger study is required to better confirm the data.

Flow cytometry is a technique often used to count cells. You stain the cells with a flourescent dye and put them in the flow cytometer it shines a laser onto the cells and if they have the dye they glow and the machine detects this. Non-expressing cells do not glow.

This study highlights a risk that you need to be aware of when you read papers. In a cell sorting machine it normally looks at say 10,000 events or 10,000 cells and does this in a few seconds, so if we get the result that say 20% are type X so in sample there are 2,000 cells. This may mean that in 100,000 cells in the body there are 20,000 cells X. So we do it again after Natalizumab and there are now 10% positive cells there are 1,000 X cells in the sample. So there is a reduction in cell type X.

Now, because Natalizumab is stopping cell type Y from entering the CNS rather than stopping cell type X getting into the lymph gland rather than there being 100,000 cells in the blood there are 200,000 cells but still only 20,000 type X cells because they have not been affected. So you do the same experiment on the flow cytometer and in the 10,000 cell sample you would now only have 1,000 cells X and 10% type X cells. However, it is not cell type X that has gone down but that other cell types have gone up that is the important result.

CD62L is a molecule (a carbohydrate) that helps T cells exit the blood and travel into lymph glands. This molecule is expressed on virgin T cells that have not been activated. They are made in the thymus and then you want them to travel to the lymph glands because that is where sensitizing antigens (infectious agents) are going to be brought by the antigen presenting cells. In the lymph glands if these naive cells see their target antigen, they will divide and expand so on next encounter with the target there are lots of cells there to fight the infection. They then change their surface markers to that of an activated cell.

One thing is to down regulate CD62L and upregulate CD49d and CD44. This because when there is inflammation in tissues the blood vessels produce vascular cell adhesion molecule (VCAM) bound by CD49d and hyalouronic acid bound by CD44 as a marker to get activated cells into sites of infection. You don't want the virgin cells entering the site of infection as they have not been primed to destroy the infection and there are not many of them.

So natalizumab works by blocking CD49d on the white blood cells so that they can't bind to VCAM on the blood vessel and so they get stuck in the blood and increase in number, so it is low CD62L that is marker of how good the antibody is at stopping the CD49d cells getting into the brain and if they can't do this then there is a risk of developing PML.

So as a biomarker is perhaps does not matter in this instance but it is worth thinking about when you read papers with flow cytometry and percentages. For this reason it is often helpful to work in whole cell numbers.

If there are say 5% CD4 T regs in normal blood with 100,000 cells per microlitre but 40% T regs after Alemtuzumab is this the reason for benefit in MS?.

However this is 40% of 150 cells per microlitre after Alemtuzumab (figures are not accurate) and is there infact less regulation. As this means there were 5,000 per microlitre to start verses 60 cells after treatment and they can't control the B cells (20% of population) which have gone-up in number by 200% in the same period, so 20,000 B cells have become 40,000 B cells and T reg to B cell ratio has gone from 5,000:20,000 (1 in 4 ratio) to 60:40,000 (1 in 333ratio). Is this why we get B cell autoimmunity after alemtuzumab.

2 comments:

Great comments on flow cytometry and pitfalls associated with data interpretation!On the subject of L-selectins and PML risk: I read somewhere that when integrin - integrin receptor binding is blocked by Tysabri, selectins may help with lymphocyte trafficking into the CNS, and low selectin expression would then prevent immune response to the JC virus in the CNS. Is this true?

I am not sure there is L-selectin ligand expression on BBB vascularture(GlyCAM-1, found in the high endothelial venules of the lymph nodes, CD34, found on endothelial cells, MadCAM-1, found on endothelial cells of gut-associated lymphoid tissue), although we showed some vessels get MadCam. CNS has P-selectin

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